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- Upstream of precise disease models for better downstream decision makingPublication . Duarte, Ana; Moreira, Luciana; Ribeiro, Diogo; Bragança, José; Amaral, OlgaInborn errors of metabolism are a common cause of inherited diseases. Diseases of carbohydrate metabolism pathway include lysosomal storage diseases (LSDs), which are a significant subgroup with approximately 70 LSDs. Grouped according to their defective lysosomal proteins and pathways they are usually characterized by intralysosomal accumulation of substrate. Accumulation may occur at different levels in diverse types of cells, some of which are of difficult access. Patient, molecular, cell and tissue heterogeneity hinders the development of further therapeutic approaches. We are currently establishing human Induced Pluripotent Stem Cells (iPSCs) from fibroblasts of LSDs patients and controls. The use of disease-specific cell models, mimicking the cell-target of the specific disease, may help to appropriately study the pathogenesis as well as the therapeutics. Integrating new techniques in the work pipeline for the establishment of models may lead to more accurate models while ensuring the safeguard of the patient’s background. Advanced technologies like microarray and NGS profiling add to the traditional techniques such as Immunofluorescence, directed sequencing and conventional cytogenetics. As in the diagnosis process, we may better understand the prognosis, and contribute to cost avoidance, by combining genomic and protein profiling checkpoints in the cell-model establishment pipeline. The investment in the upstream checkpoints might prove to be helpful in ensuring the integrity of the cell models.
- A first step to open the neuronal box of Gaucher CellsPublication . Ribeiro, Diogo; Duarte, Ana; Santos, Renato; Amaral, OlgaThis work focuses on the differentiation and gene expression characterization of neural progenitor cells obtained from human induced pluripotent cells (hiPSCs) reprogrammed from type 3 GD (GD3) fibroblasts. GD3 patient fibroblasts (from an international cell bank) were cultured and reprogramed as previously described (https://doi.org/10.1016/j.scr.2019.101595). The resulting hiPSCs were differentiated into pre-neuronal cells and, at this stage, they were examined. The gene expression behavior of all neurogenesis genes (NES, MAP2 and OTX2) was similar but higher expression was observed in GD3 hiPSCs than in GD3 neural progenitor cells. With this work, we can conclude that, when working with hiPSCs in the process of creating disease-specific cell models it is most important to carry out a general gene expression characterization of the different cell lines involved in all stages.
- Preliminary characterization of lysosomal-related genes in two tay sachs variant B1 fibroblast cell linesPublication . Ribeiro, Diogo; Marisa, Encarnação; Silva, Lisbeth; Duarte, Ana Joana; Alves, Sandra; Amaral, OlgaAim: The present work focus on the NGS characterization of lysosomal-related genes in two skin fibroblast cell lines from TSD variant B1 patients with the mutation p.R178H prior to the manipulations to generate hiPSCs and NPCs. Registering the gene variants at “time zero” will allow the eventual detection of future alterations.
- Induced pluripotent stem cell line (INSAi002-A) from a Fabry Disease patient hemizygote for the rare p.W287X mutationPublication . Duarte, Ana; Ribeiro, Diogo; Santos, Renato; Moreira, Luciana; Bragança, José; Amaral, OlgaFabry Disease (FD) is a multisystemic X-linked disorder that belongs to the group of lysosomal storage disorders (LSDs). Causal mutations on alpha-galactosidase A (α-Gal A) commonly lead to abnormal protein and consequently to FD. Since it is an X-linked disease, males are primarily affected. This work describes the generation of induced Pluripotent Stem Cells (iPSCs) from skin fibroblasts from a FD patient, using non-integrative episomal vectors. Differentiation of iPSCs can be applied to generate a variety of cell types with high degree of genetic complexity that would otherwise be difficult to obtain.
- Can Cell-type specific variability be involved in a rare variant of Unverricht-Lundborg? Investigation with iPSC generated modelsPublication . Duarte, Ana Joana; Ribeiro, Diogo; Moreira, Luciana; Amaral, OlgaHomozygosity for a private synonymous mutation in the cystatin-B gene (CSTB, MIM:601145; c.66G>A; p.Q22Q) was detected in a Portuguese patient with a rare, atypical form of Unverricht-Lundborg disease (ULD, MIM #254800). This apparently silent mutation leads to mis-splicing of CSTB pre-mRNA where a normal and an abnormal transcript were detected. Using iPSCs as a source of different cell types, we intend to clarify if the observed abnormal RNA splicing is cell-type specific, and to characterise the subsequent protein mislocalization. In conclusion, we hope to be able to contribute to the understanding of cell-type specific implications in the pathogenesis of ULD.